PROJECT SUMMARY (See instructions); As iron (Fe)-related disorders are prevalent world-wide, understanding Fe homeostasis is critical to understanding human health. Fe homeostasis is regulated by hepcidin, a largely liver-derived peptide that inhibits dietary Fe absorption and macrophage Fe recycling. Hepcidin expression Is inhibited and stimulated respectively by increased erythropoietic drive and Fe overload. The first objective of this K99/R00 application was to determine the effect of transferrin (TF) on hepcidin expression in vivo. This objective has been completed. Studies during the K99 phase demonstrated that TF, an abundant serum metal-binding protein essential for Fe delivery to bone marrow for erythropoiesis, regulates hepcidin expression dependently and Independently of TF's role In erythropoiesis. These studies also demonstrated that TF Is essential for the stimulation of hepcidin expression by Fe overload; this stimulation is a likely target ofthe unidentified factor that mediates suppression of hepcidin expression by increased erythropoietic drive. The second objective of this K99/R00 application, to be completed during the ROO phase, is to identify commonalities and divergences between cellular manganese (Mn) and Fe homeostasis as a means to further dissect Fe homeostasis. I will identify the means by which TF saturated with Fe, Mn and other metals similarly and differentially regulate hepatocyte gene expression and determine if TF plays a role In cellular Fe and Mn efflux. The third objective, also to be completed during the ROO phase, is to identify novel factors required for cellular metal homeostasis. As high Mn levels are toxic to many cell lines, I will alter cellular gene expression with shRNA and cDNA libraries, select for cells resistant to high Mn levels and Identify mutant lines of interest by 54Mn and 55Fe transport assays. From these two objectives, I will establish an Independent focus divergent from my mentor's, the role of Iron in hematologic disorders. This proposal reflects the overall trend in my research the study of mammalian metal homeostasis and its relation to human health and diseaseand will lead to a paradigm of metal homeostasis from which novel treatments for metal-related disorders can be designed.